Research Project: Biology and Management of Dipteran Pests of Livestock and Other Animals

Location: Arthropod-borne Animal Diseases Research

2021 Annual Report

Objectives
Objective 1: Conduct surveillance and evaluate the effect of nocturnal dipteran pests on dairy cattle and estimate their impact on production by quantifying defensive behaviors. These studies are intended to inform development of insect management strategies. Objective 2: Develop new and improved strategies to reduce transmission risk between livestock and biting midges that are vectors of Bluetongue and Epizootic Hemorrhagic Disease. Objective 2A: Describe species abundance, diversity, and habitat associations of larval and adult Culicoides communities collected on agricultural and wild sites in Northeastern Kansas which will facilitate improved, more targeted control strategies for midges. Objective 3: Determine the risk of bacterial pathogen transmission by house flies and develop strategies to mitigate pathogen transmission.

Approach
Among insects, Dipteran species that significantly impact livestock and human health. The studies presented here focus on three key dipteran pests: mosquitoes, biting midges and house flies. Hematophagous mosquitoes and biting midges cause direct damage to the host during blood feeding, while vector species transmit disease agents that cause morbidity and mortality. House flies are nuisance pests to humans and livestock, and annoyance is exacerbated when animals are confined in high density. Being filth-associated, house flies also disseminate and transmit a wide variety of microbes, including pathogenic or antimicrobial-resistant species, especially in operations with poor waste management. The common purpose of the proposed project is to understand key components of the host-pathogen-vector cycle to: (1) estimate pest impact on livestock and/or human health, (2) inform mitigation and management strategies for reducing host contact and pest populations and (3) ultimately reduce or prevent pathogen transmission. The mosquito projects will quantify fitness and economic impacts using wearable technology while also evaluating efficacy of novel management strategies. The biting midge research uses transcriptomics to explore how virus infection alters sensory perception and neurological function in midges, providing information key to developing or modifying control methods. The house fly studies utilize both next-generation sequencing and culture-based approaches to characterize the bacterial microbiome in flies collected from cattle operations across four US climate zones. Data will be used to perform risk assessment, pathogen and antimicrobial-resistance surveillance and to identify biotic and management variables associated with changes in the fly associated microbial community.

Progress Report
Objective 1: Research continued to opportunistically collect dairy cattle behavior data using the eartag monitoring system used by the dairy. Seasonal collections during insect free times (early spring, late fall, and winter) provide patterns of behaviors that are compared to spring, summer, and fall) when biting insects are present and the animals exhibit defensive behaviors. Combining the defensive behavioral data with insect trapping data (insect abundance) adjacent to the pens, a clear change in cattle behavior is present during weeks of high insect abundance, compared to weeks when low temperatures or storms limit insect movement to the traps and cows. The nocturnal biting insects clearly alter the total time spent sleeping and ruminating (low energy activities) at night and increase the mid and high energy activities. However, the impact of these behavioral changes has not been linked to milk production to date. Objective 2: Research on the effect of Bluetongue virus (BTV) on gene expression in female midges (Culicoides sonorensis) was performed in spring-summer 2021. Female midges were either fed a virus treatment bloodmeal with BTV, a control blood meal with just virus media, or sugar and the experiment had three biological replicates. RNA was sequenced by a process called RNAseq and the counts of RNA were compared across the conditions and normalized across the replicates. Informatic analysis of the RNAseq data is being performed late 2021. The aim is to identify genes, gene families, and gene networks that are differentially expressed across these three treatments. These differentially expressed genes give insight into the midge’s molecular phenotype as it responds to the virus infection, and can help us elucidate pathways of viral defense, vector competence, track the host response to viral dissemination, and understand the effect of BTV infection on midge neurosensory function (as has been seen previously with infection with another orbivirus, EHDV). Objective 2, 2A: This research subobjective was added in fiscal year (FY) 21 to incorporate additional research areas added to the project. Field sites were established at agriculturally associated and wild sites grazed by different large herbivores (cattle, bison, sheep, deer) at the end of FY 20 into FY 21. At each site, mud is being collected for larval emergence and identification and Centers for Disease Control and Prevention (CDC) miniature light traps are being deployed to collect adult biting midges. All midges are being identified to species and, for adults, physiological status. Additional environmental data, such as site moisture and animal presence, are being documented. Objective 3: Molecular investigations of bacterial carriage by house flies in U.S. cattle operations continued in FY 21. From July-October 2020, female house flies (n=10 to 12) and manure (n=5) samples were collected from Oklahoma, Kansas and Texas dairy farms and feedlots. DNA was extracted from whole flies and 16S rRNA genes were sequenced to categorize and quantify bacterial communities. At the time of this report, fly bacterial communities from the beef cattle farms have been analyzed. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the most dominant and prevalent bacterial phyla associated with flies from feedlots. Both geographic location and sampling month significantly affected the abundance of Firmicutes, Bacteroidetes, and Actinobacteria in flies. Highly prevalent taxa included potential pathogens of humans and livestock such as Staphylococcus (associated with cutaneous lesions and cattle diseases such as mastitis), Escherichia (associated with foodborne illness), and Moraxella (the causative agent of infectious bovine keratoconjunctivitis, or pink eye), along with several others. Bacterial species richness increased from northern to southern geographic regions and also varied by date (lowest in September, highest in October). Bacterial community compositions of individual flies from the same sampling date and geographic location were more similar indicating the pronounced effect of both variables on fly carriage of bacteria. In 2021, house flies are being collected from dairy farms in eastern states from May-August 2021 for additional molecular analyses of bacterial communities. Culture-based studies of antimicrobial-resistant (AMR) bacterial carriage by house flies also continued in 2020-2021. On 4 dates in summer 2020, male and female house flies were collected from beef cattle operations in four Kansas counties, along with manure, feed, and water samples. Flies and environmental samples were cultured on MacConkey agar to enumerate enteric bacteria. Cultures were replica plated on antibiotic media (tetracycline, florfenicol, or enrofloxacin) to determine single and multi-drug resistance in isolates. Fly sex and collection date significantly affected the abundance of bacteria in flies. Within each site, and overall, female flies carried a higher bacterial abundance than males. Male fly bacterial abundance was affected by collection date at two sites. Bacterial abundance in environmental samples varied among beef operations. Flies carried the greatest variability in bacterial morphotypes, and many were similar to those observed in environmental samples. Species identification of AMR isolates is ongoing, and species shared among flies and environmental samples will be subjected to whole genome sequencing in 2022 in order to determine the genetic nature of AMR in the isolates.

Accomplishments
1. Female house flies harbor bacterial communities that reflect their surrounding environment. House flies are ubiquitous pests that occupy a variety of ecological niches, such as urban environments, suburban locales, and animal agriculture facilities. Female house flies visit microbe-rich substrates in these habitats so they can feed and deposit eggs, and in this process acquire bacteria. Researchers in Manhattan, Kansas, in collaboration with Kansas State University, investigated the bacterial communities carried in the gut of field-caught female house flies from three different environments: an agricultural habitat (cattle farm), a mixed habitat (business with nearby agriculture) and an urban habitat (restaurant dumpsters). The team collected flies in May, June, and July and used a next-generation sequencing approach to characterize the bacterial species carried within the flies. Bacterial communities were most similar among flies collected from the same habitat, and within each habitat some types of bacteria that flies carried changed over time. Flies from the cattle farm carried a more diverse bacterial community than those from the urban or mixed habitats. Irrespective of habitat, flies carried potential human and animal pathogens, but the types of pathogens were strongly influenced by the environmental niche. This study adds to the growing body of evidence implicating house flies in harboring and disseminating bacteria of medical and veterinary importance. Furthermore, because the bacteria carried by flies seem to represent a snapshot bacteria found in their surrounding environment, we can use flies as sentinels to monitor existing and emergent threats to human and animal health.

2. House flies carry antimicrobial-resistant and multidrug-resistant bacteria at Kansas cattle operations. Antimicrobial resistance (AMR) is a top priority for livestock producers due to implications to both animal and human health. AMR usually results from repeated use of antimicrobials to control or prevent disease in production animals. House flies are prevalent in production settings due to open access to manure, which serves as an ideal developmental substrate for fly larvae. Animals shed AMR bacteria in their manure, and adult house flies acquire and disseminate bacteria throughout operations. Thus, house flies may be integral in the persistence and movement of bacteria and AMR at cattle operations. ARS scientists in Manhattan, Kansas, along with collaborators from Kansas State University collected house flies from Kansas dairy and beef facilities for five alternating weeks in summer 2019. Bacteria associated with manure, known as coliforms, were enumerated and were screened for antimicrobial resistance to the drugs florfenicol, enrofloxacin, ceftiofur, and ampicillin. Overall, female flies carried more bacteria than males. Approximately 46% of coliforms from flies were AMR, and 13% were multi-drug resistant (MDR). 73% of flies carried at least one AMR coliform, and 24% of flies carried MDR coliforms. Most MDR bacteria were resistant to tetracycline with ampicillin and/or florfenicol. Only female flies carried enrofloxacin- or ceftiofur-resistant bacteria, and only female flies from beef operations carried enrofloxacin resistance. One coliform isolated from female fly collected at a beef cattle operation was resistant to all five antibiotics. Our results show that house flies play a significant role in harboring and transmitting AMR and MDR bacteria in confined cattle operations and fly management should be paramount in AMR mitigation strategies.

3. Precision agriculture for intensive animal production. ARS researchers in Manhattan, Kansas, in collaboration with Kansas State University developed a feeding station that customizes and monitors the feed of individual animals. A unique identifier (ear tag, collar, etc.) on the animal (cow, dog, etc.) triggers the system to allow the animal to feed. The system records how much food or water the animal consumes (by weight). The system can also provide individualized treatments for endo- or ectoparasites by mixing it into the feed and the system insures all the animals receive treatment. Eventually, artificial intelligence can be used to monitor consumption and detect significant changes in feeding behavior (consumption and timing) to inform producers of a problem with an individual animal in the herd. Although the system can be used by the general public to feed dogs and cats or commercially in dairies or piggeries, the system was designed to be used in remote isolated environments such as with pastured beef cattle because it can be powered by a solar panel and used to monitor difficult to observe animals.

4. House flies can harbor SARS-CoV-2 but do not transmit infectious virus. SARS-CoV-2 is a recently emerged coronavirus that is the causative agent of the global COVID-19 pandemic. The virus is highly contagious and is usually transmitted to new hosts via the respiratory route through aerosols, or after contact with items contaminated by infected persons. House flies transmit various pathogens to humans and animals as mechanical vectors, however the house fly’s role in SARS-CoV-2 transmission had not yet been explored. ARS scientists in Manhattan, Kansas, along with collaborators from Kansas State University determined whether house flies could acquire and transmit the SARS-CoV-2. virus. Flies were exposed to SARS-CoV-2-spiked culture media or 10% milk substrates and tested for virus at either 4 or 24 hours after exposure. All flies exposed to SARS-CoV-2- inoculated media or milk were positive for viral RNA at 4 hours and 24 hours post-exposure. However, infectious virus was detected only from the flies exposed to virus-spiked milk. In a second experiment, flies were exposed to a substrate containing virus for 24 hours (as well as positive and negative control substrates). Flies then were transferred to a clean container with naive substrate, and after 4 and 24 hours flies, container swabs and the naive substrate were removed and tested for SARS-CoV-2. The nucleic acid from the virus (e.g., viral RNA) was detected in environmental samples (swabs, naïve substrates) after contact with SARS-CoV-2 exposed flies, but no infectious virus was recovered. Therefore, under laboratory conditions using high amounts of virus, house flies were able to acquire and harbor infectious SARS-CoV-2 for up to 24 hours post-exposure but they were not able to mechanically transmit infectious virus, only the virus RNA, which is not infectious. While house flies likely do not play an important role in SARS-CoV-2 transmission, field-trapped house flies could potentially be used for surveillance of virus in the environment within which they are captured.

Review Publications
McGregor, B.L., Giordano, B.V., Runkel, A.E., Nigg, H.N., Nigg, H., Burkett-Cadena, N.D. 2020. Comparison of the effect of insecticides on bumble bees (Bombus impatiens) and mosquitoes (Aedes aegypti and Culex quinquefasciatus) by standard mosquito research methods. Journal of Economic Entomology. 114(1):24-32. https://doi.org/10.1093/jee/toaa282.
McGregor, B.L., Connelly, C., Kenney, J.L. 2021. Infection, dissemination, and transmission potential of North American Culex quinquefasciatus, Culex tarsalis, and Culicoides sonorensis for Oropouche virus. Viruses. 113(2):226. https://doi.org/10.3390/v13020226.
Balaraman, V., Drolet, B.S., Mitzel, D.N., Wilson, W.C., Owens, J.L., Gaultiero, N.N., Meekins, D.A., Bold, D., Trujillo, J.D., Noronha, L.E., Richt, J.A., Nayduch, D. 2021. Mechanical transmission of SARS-CoV-2 by house flies. Parasites & Vectors. 14:214. https://doi.org/10.1186/s13071-021-04703-8.
Turell, M.J., Cohnstaedt, L.W., Wilson, W.C. 2020. Effect of environmental temperature on the ability of Culex tarsalis and Aedes taeniorhynchus (Diptera: Culicidae) to transmit Rift Valley fever virus. Vector-Borne and Zoonotic Diseases. 20(6):454-460. https://doi.org/10.1089/vbz.2019.2554.
Thomson, J.L., Cernicchiaro, N., Zurek, L., Nayduch, D. 2021. Cantaloupe facilitates Salmonella Typhimurium survival within and transmission among adult house flies (Musca domestica L.). Foodborne Pathogens and Disease. 18(1):49-55. https://doi.org/10.1089/fpd.2020.2818.
Yang, Q., Yi, C., Vajdi, A., Cohnstaedt, L.W., Wu, H., Guo, X., Scoglio, C. 2020. Short-term and long-term modeling of the COVID-19 epidemic in the Hubei Province. Journal of Infectious Diseases and Therapy. 5:563-574. https://doi.org/10.1016/j.idm.2020.08.001.
McGregor, B.L., Blackburn, J.K., Wisely, S.M., Burkett-Cadena, N.D. 2021. Culicoides (Diptera: Ceratopogonidae) communities differ between a game preserve and nearby natural areas in northern Florida. Journal of Medical Entomology. 58(1):450-457. https://doi.org/10.1093/jme/tjaa152.
Baliota, G.V., Athanassiou, C.G., Cohnstaedt, L.W. 2021. Response of phosphine-resistant and -susceptible Lasioderma serricorne adults to different light spectra. Journal of Stored Products Research. 92:101808. https://doi.org/10.1016/j.jspr.2021.101808.
McGregor, B.L., Connelly, C. 2021. A review of the control of Aedes aegypti (Diptera: Culicidae) in the continental United States. Journal of Medical Entomology. 58(1):10-25. https://doi.org/10.1093/jme/tjaa157.
Olafson, P.U., Askoy, S., Attardo, G.M., Buckmeier, B.G., Chen, X., Coates, C.J., Davis, M.C., Dykema, J., Emrich, S., Friedrich, M., Holmes, C.J., Ioannidis, P., Jansen, E.N., Jennings, E.M., Lawson, D., Martinson, E.O., Maslen, G.L., Meisel, R.P., Murphy, T.D., Nayduch, D., Nelson, D.R., Oyen, K.J., Raszick, T., Ribeiro, J.M., Robertson, H.M., Rosendale, A.J., Sackton, T.B., Saelao, P., Swiger, S.L., Sze, S., Tarone, A., Taylor, D.B., Warren, W.C., Waterhouse, R.M., Weirauch, M.T., Werren, J.H., Wilson, R.K., Zdobnov, E.M., Benoit, J.B. 2021. The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control. BMC Biology. 19:41. https://doi.org/10.1186/s12915-021-00975-9.
Ghosh, A., Jasperson, D.C., Cohnstaedt, L.W., Brelsfoard, C. 2019. Transinfection of Culicoides sonorensis biting midge cell lines with Wolbachia pipientis. Vector-Borne and Zoonotic Diseases. 12:483. https://doi.org/10.1186/s13071-019-3716-0.